| Abstract
| - Overexpression of Bcl-2, an anti-apoptotic oncoprotein, is commonly observed in a variety ofhuman malignancies and is associated with resistance to chemotherapy and radiotherapy. Although theprecise mechanism of Bcl-2 action remains elusive, current evidence indicates that Bcl-2 inhibits apoptosisby binding and inhibiting pro-apoptotic molecules such as Bax. Therefore, agents that disrupt the abilityof Bcl-2, or other anti-apoptotic molecules, to bind to pro-apoptotic molecules may have therapeutic value.Several studies have shown that the BH3 domains of Bcl-2 and Bax are critically important for Bax/Bcl-2 heterodimerization. In this report, we designed and synthesized peptides based on the BH3 domainsof three distinct Bcl-2 family members, Bcl-2, Bax and Bad. In vitro interaction assays were used tocompare the abilities of the different peptides to inhibit Bax/Bcl-2 and Bax/Bcl-xL heterodimerization, aswell as Bcl-2 and Bax homodimerization. Bax BH3 peptide (20-amino acids) potently inhibited bothBax/Bcl-2 and Bax/Bcl-xL interactions, exhibiting IC50 values of 15 and 9.5 μM, respectively. The BadBH3 peptide (21 amino acids) was slightly more potent than Bax BH3 at inhibiting Bax/Bcl-xL but failedto disrupt Bax/Bcl-2. Bcl-2 BH3 peptide (20-amino acids) was inactive toward Bax/Bcl-2 and had onlya weak inhibitory effect on Bax/Bcl-xL heterodimerization. All three BH3 peptides failed to significantlyinhibit homodimerization of Bcl-2 or Bax. Consistent with its ability to disrupt Bax/Bcl-2 heterodimerization, Bax BH3 peptide was able to overcome Bcl-2 overexpression and induce cytochrome c releasefrom mitochondria of Bcl-2-overexpressing Jurkat T leukemic cells. Bad BH3 peptide, while potentlyinducing cytochrome c release in wild-type Jurkat cells, only partially overcame the effects of Bcl-2overexpression. Bcl-2 BH3 failed to induce cytochrome c release, even in wild-type cells. Delivery of theBax BH3 and Bad BH3 peptides into wild-type Jurkat cells induced comparable levels of cell death. Incells overexpressing Bcl-2, the potency of Bax BH3 peptide was similar to that seen in wild-type cells,while the efficacy of Bad BH3 peptide was reduced. By contrast, in Bcl-xL-overexpressing cells, BadBH3 exhibited greater cell-killing activity than Bax BH3. The Bcl-2 BH3 peptide and a mutant Bax BH3peptide had no appreciable effect on Jurkat cells. Together, our data suggest that agents based on the BaxBH3 domain may have therapeutic value in cancers overexpressing Bcl-2, while agents based on theBH3 domain of Bad may be more useful for tumors overexpressing Bcl-xL.
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